Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability

Manganese peroxidase (MnP) produced by Phanerochaete chrysosporium, which c atalyzes the oxidation of Mn2+ to Mn3+ by hydrogen peroxide, was shown to b e susceptible to thermal inactivation due to the loss of calcium [Sutherlan d, G. R. J.; Aust, S. D. Arch. Biochem. Biophys. 1996, 332, 128-134]. The r ecombinant enzyme, lacking glycosylation, was found to be more susceptible [Nie, G.; Reading, N. S.; Aust, S. D. Arch. Biochem. Biophys. 1999, 365, 32 8-334]. On the basis of the properties and structure of peanut peroxidase, we have engineered a disulfide bond near the distal calcium binding site of MnP by means of the double mutation A48C and A63C. The mutant enzyme had a ctivity and spectral properties similar to those of native, glycosylated Mn P. The thermostabilities of native, recombinant, and mutant MnP were studie d as a function of temperature and pH. MnPA48C/A63C exhibited kinetics of i nactivation similar to that of native MnP. The addition of calcium decrease d the rate of thermal inactivation of the enzymes, while EGTA increased the rate of inactivation. Thermally treated MnPA48C/A63C mutant was shown to c ontain one calcium, and it retained a percentage of its original manganese oxidase activity; native and recombinant MnP were inactivated by the remova l of calcium from the protein.